The sense of smell plays an important role in our quality of life. However, the mechanisms governing the processing and representation of olfactory information in the brain are not well understood. In mammals, the olfactory bulb is a critical brain region responsible for the initial processing of olfactory information. Much of our current understanding of olfactory bulb function is based on previous studies using in vitro brain slices or acute preparations of anesthetized animals. While these approaches have provided valuable insight, much less is known about odor coding in awake, behaving animals. For example, what are the dynamics of odor representations in the same animal over long time scales (i.e. days, weeks, months) and how is odor coding in the olfactory bulb shaped by experience and learning in awake, behaving animals? To address these questions, we propose an experimental strategy using chronic 2-photon calcium imaging to study odor-evoked activity in the olfactory bulbs of awake mice. Specific Aim 1 proposes the development of an approach for the selective expression of the genetically-encoded calcium indicator GCaMP3 in principal (mitral/tufted) cells or local interneurons using a Cre-dependent, adeno-associated virus system. We hypothesize that this approach will provide single cell resolution of action potential-dependent calcium signals in large populations of neural ensembles that can be imaged chronically in awake, head fixed mice. Specific Aim 2 proposes imaging experiments to determine how odor-evoked responses of mitral cells and interneurons (granule cells) differ between the awake and anesthetized state. We will also use chronic imaging in awake animals to determine whether patterns of odor-evoked activity in neural ensembles are stable or dynamic over days of repeated testing. These experiments will provide new insight into the nature of odor coding in the awake brain. PUBLIC HEALTH RELEVANCE: Revealing how sensory information is processed and encoded by neural circuits is critical for understanding brain function. Here we propose a novel optical imaging approach to study how olfactory information is represented by large neuronal ensembles in awake mice.